Squamous cell carcinomas of the skin (SCC) are a leading cause of death in organ transplant recipients and treatment of non-melanoma skin cancers, of which SCC is the second most frequent type, account for 4.5% of all Medicare cancer costs. The incidence and malignancy of SCC are greatly increased in patients with decreased T cell function, suggesting a role for the immune system in controlling these tumors. T cells are found within SCC but fail to control tumor growth. Preliminary results are presented that SCC evade the immune system by two newly identified mechanisms. First, by failing to express E-selectin on tumor vessels, SCC evade the population of antigen-experienced skin homing T cells most capable of recognizing the tumor. Second, by recruiting FOXP3+ regulatory T cells (Treg), SCC create a local environment of immune suppression around the tumor. Imiquimod, a TLR7 agonist effective in the treatment of skin cancers, neutralizes both of these defenses, inducing expression of E-selectin on tumor vessels, restoring the ability of CLA+ skin homing T cells to enter the tumor and reducing the % FOXP3+ Treg to levels found in normal skin. Studies described in this proposal focus on determining how SCC inhibit vascular E-selectin and recruit Treg with the goal of developing novel agents for the treatment of SCC and their premalignant precursor lesions, actinic keratoses (AK). Additional studies focus on determining the mechanisms by which imiquimod induces vascular E-selectin and restores T cell homing, with the goal of developing agents that can treat established SCC without the risk of widespread immune stimulation that is a concern with TLR agonists such as imiquimod. Hypotheses are presented that nitric oxide (NO) produced by dendritic cells (DC) within SCC inhibit vascular E-selectin and that recruitment of NO-producing DC and Treg both occur via CCR2. Proposed studies investigate if inhibition of CCR2 function, induction of E-selectin on tumor vessels and inhibition of Treg recruitment can enhance the immunologic response to SCC. These questions will be investigated first in vitro and then in immunodeficient mice grafted with human SCC tumors. Lastly, studies are proposed to determine if aberrant homing and Treg recruitment also occur in AK. If so, therapies that induce E-selectin and inhibit recruitment of Treg may be effective in the treatment of these lesions, inducing their immunologic destruction before they progress to SCC. Novel therapies will be evaluated in vitro and in mice grafted with human AK. These studies address two long-term interests of NIAMS: study of the skin as an immune organ and study of the role of the immune system in the development and treatment of skin cancer.